Bottom Line:
Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

ABSTRACTStriated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

RSOB140041F3: TK contains atypical residues in catalytic motifs. (a) Distribution of residues in position 2 of the VAIK motif and position 1 of the DFG motif in protein kinases of the human kinome. The classification of kinases and pseudokinases was taken from [32]. TK, the only human kinase containing an EFG motif, is misclassified as active kinase due to previous reports of catalysis [10,13,16]. Other kinases with deviant catalytic motifs are: CASK (GFG motif); ATR (xMxK motif); LMR2, NEK8 and RIPK1 (IxK); and DNAPK, FRAP and SMG1 (LxK). (b) Commonly occurring residues in the VAIK and DFG motifs of titin kinases from vertebrate (V) and invertebrate titin-like kinases (Inv). (Sequences for representative kinases of each group are given in the electronic supplementary material, figure S5.)

Mentions:
In the DFG motif, aspartate chelates the magnesium ion that commonly coordinates the β-phosphate of ATP. This residue is conserved across most members of the protein kinase-like superfamily [29]. Although glutamate and aspartate are chemically similar, this substitution is sufficient to inactivate phospho-transfer in kinases [30,31]. In the human kinome [32], the deviant residues found in TK are extremely rare among active kinases (figure 3). TK is the only known human kinase that contains glutamate instead of aspartate in the DFG motif, while ATR kinase is the only other human kinase containing a methionine in position 2 of the VAIK motif (but its level of activity is unclear). By contrast, among pseudokinases there is no marked selection for given residues in position 2 of the VAIK motif or aspartate in the DFG signature. Thus, the YMAK/EFG signatures clearly point to irregular catalysis in TK.Figure 3.

RSOB140041F3: TK contains atypical residues in catalytic motifs. (a) Distribution of residues in position 2 of the VAIK motif and position 1 of the DFG motif in protein kinases of the human kinome. The classification of kinases and pseudokinases was taken from [32]. TK, the only human kinase containing an EFG motif, is misclassified as active kinase due to previous reports of catalysis [10,13,16]. Other kinases with deviant catalytic motifs are: CASK (GFG motif); ATR (xMxK motif); LMR2, NEK8 and RIPK1 (IxK); and DNAPK, FRAP and SMG1 (LxK). (b) Commonly occurring residues in the VAIK and DFG motifs of titin kinases from vertebrate (V) and invertebrate titin-like kinases (Inv). (Sequences for representative kinases of each group are given in the electronic supplementary material, figure S5.)

Mentions:
In the DFG motif, aspartate chelates the magnesium ion that commonly coordinates the β-phosphate of ATP. This residue is conserved across most members of the protein kinase-like superfamily [29]. Although glutamate and aspartate are chemically similar, this substitution is sufficient to inactivate phospho-transfer in kinases [30,31]. In the human kinome [32], the deviant residues found in TK are extremely rare among active kinases (figure 3). TK is the only known human kinase that contains glutamate instead of aspartate in the DFG motif, while ATR kinase is the only other human kinase containing a methionine in position 2 of the VAIK motif (but its level of activity is unclear). By contrast, among pseudokinases there is no marked selection for given residues in position 2 of the VAIK motif or aspartate in the DFG signature. Thus, the YMAK/EFG signatures clearly point to irregular catalysis in TK.Figure 3.

Bottom Line:
Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

ABSTRACTStriated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.